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Specimen-specific drift of densities defines distinct subclasses of extracellular vesicles from human whole saliva.

ABSTRACT: Extracellular vesicles (EVs) in body fluids constitute heterogenous populations, which mirror their diverse parental cells as well as distinct EV-generation pathways. Various methodologies have been proposed to differentiate EVs in order to deepen the current understanding of EV biology. Equilibrium density-gradient centrifugation has often been used to separate EVs based on their buoyant densities; however, the standard conditions used for the method do not necessarily allow all EVs to move to their equilibrium density positions, which complicates the categorization of EVs. Here, by prolonging ultracentrifugation time to 96 h and fractionating EVs both by floating up or spinning down directions, we allowed 111 EV-associated protein markers from the whole saliva of three healthy volunteers to attain equilibrium. Interestingly, the determined buoyant densities of the markers drifted in a specimen-specific manner, and drift patterns differentiated EVs into at least two subclasses. One class carried classical exosomal markers, such as CD63 and CD81, and the other was characterized by the molecules involved in membrane remodeling or vesicle trafficking. Distinct patterns of density drift may represent the differences in generation pathways of EVs.

SUBMITTER: Yamamoto S

PROVIDER: S-EPMC8032098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations. The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase. The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.

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Specimen-specific drift of densities defines distinct subclasses of extracellular vesicles from human whole saliva.

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